搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Induced pluripotent stem (iPS) cells are being used increasingly to complement their embryonic counterparts to understand and develop the therapeutic potential of pluripotent cells. Our objectives were to identify an efficient cardiac differentiation protocol for human iPS cells as monolayers,and demonstrate that the resulting cardiac progenitors could provide a therapeutic benefit in a rodent model of myocardial infarction. Herein,we describe a 14-day protocol for efficient cardiac differentiation of human iPS cells as a monolayer,which routinely yielded a mixed population in which over 50% were cardiomyocytes,endothelium,or smooth muscle cells. When differentiating,cardiac progenitors from day 6 of this protocol were injected into the peri-infarct region of the rat heart; after coronary artery ligation and reperfusion,we were able to show that human iPS cell-derived cardiac progenitor cells engrafted,differentiated into cardiomyocytes and smooth muscle,and persisted for at least 10 weeks postinfarct. Hearts injected with iPS-derived cells showed a nonsignificant trend toward protection from decline in function after myocardial infarction,as assessed by magnetic resonance imaging at 10 weeks,such that the ejection fraction at 10 weeks in iPS treated hearts was 62%±4%,compared to that of control infarcted hearts at 45%±9% (Ptextless0.2). In conclusion,we demonstrated efficient cardiac differentiation of human iPS cells that gave rise to progenitors that were retained within the infarcted rat heart,and reduced remodeling of the heart after ischemic damage. View Publication

The breakthrough in derivation of human-induced pluripotent stem cells (hiPSCs) provides an approach that may help overcome ethical and allergenic challenges posed in numerous medical applications involving human cells,including neural stem/progenitor cells (NSCs). Considering the great potential of NSCs in targeted cancer gene therapy,we investigated in this study the tumor tropism of hiPSC-derived NSCs and attempted to enhance the tropism by manipulation of biological activities of proteins that are involved in regulating the migration of NSCs toward cancer cells. We first demonstrated that hiPSC-NSCs displayed tropism for both glioblastoma cells and breast cancer cells in vitro and in vivo. We then compared gene expression profiles between migratory and non-migratory hiPSC-NSCs toward these cancer cells and observed that the gene encoding neuronal nitric oxide synthase (nNOS) was down-regulated in migratory hiPSC-NSCs. Using nNOS inhibitors and nNOS siRNAs,we demonstrated that this protein is a relevant regulator in controlling migration of hiPSC-NSCs toward cancer cells,and that inhibition of its activity or down-regulation of its expression can sensitize poorly migratory NSCs and be used to improve their tumor tropism. These findings suggest a novel application of nNOS inhibitors in neural stem cell-mediated cancer therapy. View Publication

Addiction is known to occur through the consumption of substances such as pharmaceuticals,illicit drugs,food,alcohol and tobacco. These addictions can be viewed as drug addiction,resulting from the ingestion of chemical substances contained in them. Multiple neural networks,including the reward system,anti‐reward/stress system and central immune system in the brain,are believed to be involved in the onset of drug addiction. Although various compound evaluations using microelectrode array (MEA) as an in vitro testing methods to evaluate neural activities have been conducted,methods for assessing addiction have not been established. In this study,we aimed to develop an in vitro method for assessing the addiction of compounds,as an alternative to animal experiments,using human iPS cell‐derived dopaminergic neurons with MEA measurements. MEA data before and after chronic exposure revealed specific changes in addictive compounds compared to non‐addictive compounds,demonstrating the ability to estimate addiction of compound. Additionally,conducting gene expression analysis on cultured samples after the tests revealed changes in the expression levels of various receptors (nicotine,dopamine and GABA) due to chronic administration of addictive compounds,suggesting the potential interpretation of these expression changes as addiction‐like responses in MEA measurements. The addiction assessment method using MEA measurements in human iPS cell‐derived dopaminergic neurons conducted in this study proves effective in evaluating addiction of compounds on human neural networks. View Publication

Because neurons are difficult to obtain from humans,generating functional neurons from human induced pluripotent stem cells (hiPSCs) is important for establishing physiological or disease-relevant screening systems for drug discovery. To examine the culture conditions leading to efficient differentiation of functional neural cells,we investigated the effects of oxygen stress (2% or 20% O2) and differentiation medium (DMEM/F12:Neurobasal-based [DN] or commercial [PhoenixSongs Biologicals; PS]) on the expression of genes related to neural differentiation,glutamate receptor function,and the formation of networks of neurons differentiated from hiPSCs (201B7) via long-term self-renewing neuroepithelial-like stem (lt-NES) cells. Expression of genes related to neural differentiation occurred more quickly in PS and/or 2% O2 than in DN and/or 20% O2,resulting in high responsiveness of neural cells to glutamate,N-methyl-d-aspartate (NMDA),α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA),and (S)-3,5-d... View Publication

PURPOSE Demonstrate a novel manufacturing method to generate extracellular matrix scaffolds from cardiac fibroblasts (CF-ECM) as a therapeutic mesenchymal stem cell-transfer device. MATERIALS AND METHODS Rat CF were cultured at high-density (˜1.6×10(5)/cm(2)) for 10-14 days. Cell sheets were removed from the culture dish by incubation with EDTA and decellularized with water and peracetic acid. CF-ECM was characterized by mass spectrometry,immunofluorescence and scanning electron microscopy. CF-ECM seeded with human embryonic stem cell derived mesenchymal stromal cells (hEMSCs) were transferred into a mouse myocardial infarction model. 48 hours later,mouse hearts were excised and examined for CF-ECM scaffold retention and cell transfer. RESULTS CF-ECM scaffolds are composed of fibronectin (82%),collagens type I (13%),type III (3.4%),type V (0.2%),type II (0.1%) elastin (1.3%) and 18 non-structural bioactive molecules. Scaffolds remained intact on the mouse heart for 48 hours without the use of sutures or glue. Identified hEMSCs were distributed from the epicardium to the endocardium. CONCLUSIONS High density cardiac fibroblast culture can be used to generate CF-ECM scaffolds. CF-ECM scaffolds seeded with hEMSCs can be maintained on the heart without suture or glue. hEMSC are successfully delivered throughout the myocardium. View Publication

The intestinal epithelium of human infants is developmentally immature compared to that of adults. Exactly how this immaturity affects key epithelial functions and their interactions with nearby immune cells remains an understudied area of research,partly due to limited access to non-diseased infant gut tissues. Human intestinal organoids,or “mini guts” generated from tissue stem cells,are promising models for investigating intestinal biology and disease mechanisms. These three-dimensional structures closely mimic their tissue of origin,including cellular physiology and genetics. We have also previously shown that neonatal Th17 cells represent a distinct cell population with a cytokine profile skewed toward IL-22 production rather than IL-17A,as seen in adult Th17 cells. In this study,we sought to model the impact of neonatal-derived Th17 cytokine,namely IL-22 and the intestinal epithelium using infant-derived ileal enteroids. We generated enteroids from ileal biopsies from infants (< 6 months old) and cultured them for seven days with standard organoid growth media,organoid media supplemented with conditioned media from cord-blood-derived Th17 cells,or media supplemented with recombinant IL-22. We assessed morphological changes and conducted transcriptomics profiling via RNAseq. Exposing enteroids to neonatal Th17-cells-derived conditioned media led to enhanced growth,maturation,and differentiation as compared to control media. These effects were ablated when an IL-22 neutralizing antibody was used,while conversely,supplementing with recombinant IL-22 mimicked the Th17 effects,increasing intestinal epithelial cell proliferation and inducing marked differentiation of secretory cells. Our transcriptomic profiling similarly demonstrated significant changes in response to IL-22 with downregulation of Wnt and Notch signaling and upregulation of immune pathways,particularly interferon signaling. The transcriptomic data also suggested that IL-22 treatment led to changes in cell type composition with an increase in stem- and progenitor cells at the expense of enterocytes. Taken together,our data suggests that early-life intestinal development is likely influenced by IL-22-dependent crosstalk between the infant epithelium and exposure to neighboring Th17 cells. This promotes epithelial cell maturation and immune readiness,reflected at both the morphological and molecular levels. Our work also provides a relevant framework for studying healthy infant gut development,which can be further leveraged to examine early-life gastrointestinal disorders,model complex human disease,and therapeutic testing while reducing reliance on animal models. View Publication

BACKGROUND In colorectal carcinoma (CRC),activation of the Raf/MEK/ERK signaling pathway is commonly observed. In addition,the commonly used 5FU-based chemotherapy in patients with metastatic CRC was found to enrich a subpopulation of CD26(+) cancer stem cells (CSCs). As activation of the Raf/MEK/ERK signaling pathway was also found in the CD26(+) CSCs and therefore,we hypothesized that an ATP-competitive pan-Raf inhibitor,Raf265,is effective in eliminating the cancer cells and the CD26(+) CSCs in CRC patients. METHODS HT29 and HCT116 cells were treated with various concentrations of Raf265 to study the anti-proliferative and apoptotic effects of Raf265. Anti-tumor effect was also demonstrated using a xenograft model. Cells were also treated with Raf265 in combination with 5FU to demonstrate the anti-migratory and invasive effects by targeting on the CD26(+) CSCs and the anti-metastatic effect of the combined treatment was shown in an orthotopic CRC model. RESULTS Raf265 was found to be highly effective in inhibiting cell proliferation and tumor growth through the inhibition of the RAF/MEK/ERK signaling pathway. In addition,anti-migratory and invasive effect was found with Raf265 treatment in combination with 5FU by targeting on the CD26(+) cells. Finally,the anti-tumor and anti-metastatic effect of Raf265 in combination with 5FU was also demonstrated. CONCLUSIONS This preclinical study demonstrates the anti-tumor and anti-metastatic activity of Raf265 in CRC,providing the basis for exploiting its potential use and combination therapy with 5FU in the clinical treatment of CRC. View Publication

BACKGROUND Self-renewing,chemoresistant breast cancer stem cells are believed to contribute significantly to cancer invasion,migration and patient relapse. Therefore,the identification of signaling pathways that regulate the acquisition of stem-like qualities is an important step towards understanding why patients relapse and towards development of novel therapeutics that specifically target cancer stem cell vulnerabilities. Recent studies identified a role for the aryl hydrocarbon receptor (AHR),an environmental carcinogen receptor implicated in cancer initiation,in normal tissue-specific stem cell self-renewal. These studies inspired the hypothesis that the AHR plays a role in the acquisition of cancer stem cell-like qualities. RESULTS To test this hypothesis,AHR activity in Hs578T triple negative and SUM149 inflammatory breast cancer cells were modulated with AHR ligands,shRNA or AHR-specific inhibitors,and phenotypic,genomic and functional stem cell-associated characteristics were evaluated. The data demonstrate that (1) ALDH(high) cells express elevated levels of Ahr and Cyp1b1 and Cyp1a1,AHR-driven genes,(2) AHR knockdown reduces ALDH activity by 80%,(3) AHR hyper-activation with several ligands,including environmental ligands,significantly increases ALDH1 activity,expression of stem cell- and invasion/migration-associated genes,and accelerates cell migration,(4) a significant correlation between Ahr or Cyp1b1 expression (as a surrogate marker for AHR activity) and expression of stem cell- and invasion/migration-associated gene sets is seen with genomic data obtained from 79 human breast cancer cell lines and over 1,850 primary human breast cancers,(5) the AHR interacts directly with Sox2,a master regulator of self-renewal; AHR ligands increase this interaction and nuclear SOX2 translocation,(6) AHR knockdown inhibits tumorsphere formation in low adherence conditions,(7) AHR inhibition blocks the rapid migration of ALDH(high) cells and reduces ALDH(high) cell chemoresistance,(8) ALDH(high) cells are highly efficient at initiating tumors in orthotopic xenografts,and (9) AHR knockdown inhibits tumor initiation and reduces tumor Aldh1a1,Sox2,and Cyp1b1 expression in vivo. CONCLUSIONS These data suggest that the AHR plays an important role in development of cells with cancer stem cell-like qualities and that environmental AHR ligands may exacerbate breast cancer by enhancing expression of these properties. View Publication

Directed hepatocyte differentiation from human induced pluripotent stem cells (iPSCs) potentially provides a unique platform for modeling liver genetic diseases and performing drug-toxicity screening in vitro. Wilson's disease is a genetic disease caused by mutations in the ATP7B gene,whose product is a liver transporter protein responsible for coordinated copper export into bile and blood. Interestingly,the spectrum of ATP7B mutations is vast and can influence clinical presentation (a variable spectrum of hepatic and neural manifestations),though the reason is not well understood. We describe the generation of iPSCs from a Chinese patient with Wilson's disease that bears the R778L Chinese hotspot mutation in the ATP7B gene. These iPSCs were pluripotent and could be readily differentiated into hepatocyte-like cells that displayed abnormal cytoplasmic localization of mutated ATP7B and defective copper transport. Moreover,gene correction using a self-inactivating lentiviral vector that expresses codon optimized-ATP7B or treatment with the chaperone drug curcumin could reverse the functional defect in vitro. Hence,our work describes an attractive model for studying the pathogenesis of Wilson's disease that is valuable for screening compounds or gene therapy approaches aimed to correct the abnormality. In the future,once relevant safety concerns (including the stability of the mature liver-like phenotype) and technical issues for the transplantation procedure are solved,hepatocyte-like cells from similarly genetically corrected iPSCs could be an option for autologous transplantation in Wilson's disease. View Publication

Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis,biological activity,and further profiling of these compounds are described. This work resulted in the discovery of 17,GDC-0941,which is a potent,selective,orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer. View Publication

Imatinib mesylate has shown remarkable efficacy in the treatment of patients in the chronic phase of chronic myeloid leukemia. However,despite an overall significant hematological and cytogenetic response,imatinib therapy may favor the emergence of drug-resistant clones,ultimately leading to relapse. Some imatinib resistance mechanisms had not been fully elucidated yet. In this study we used sensitive and resistant sublines from a Bcr-Abl positive cell line to investigate the putative involvement of telomerase in the promotion of imatinib resistance. We showed that sensitivity to imatinib can be partly restored in imatinib-resistant cells by targeting telomerase expression,either by the introduction of a dominant-negative form of the catalytic protein subunit of the telomerase (hTERT) or by the treatment with all-trans-retinoic acid,a clinically used drug. Furthermore,we showed that hTERT overexpression favors the development of imatinib resistance through both its antiapoptotic and telomere maintenance functions. Therefore,combining antitelomerase strategies to imatinib treatment at the beginning of the treatment should be promoted to reduce the risk of imatinib resistance development and increase the probability of eradicating the disease. View Publication

Coinfection of HIV-1 patients with Plasmodium falciparum,the etiological agent of malaria,results in a raise of viral load and an acceleration of disease progression. The primary objective of this study was to investigate whether the malarial pigment hemozoin (HZ),a heme by-product of hemoglobin digestion by malaria parasites,can affect HIV-1 transmission by monocytes-derived dendritic cells (DCs) to CD4(+) T cells when HZ is initially internalized in monocytes before their differentiation in DCs. We demonstrate in this study that HZ treatment during the differentiation process induces an intermediate maturation phenotype when compared with immature and fully mature DCs. Furthermore,the DC-mediated transfer of HIV-1 is enhanced in presence of HZ,a phenomenon that may be linked with the capacity of HZ-loaded cells to interact and activate CD4(+) T cells. Altogether our findings suggest a new mechanism that could partially explain the increased HIV-1 virus production during a coinfection with P. falciparum. Understanding the multifaceted interactions between P. falciparum and HIV-1 is an important challenge that could lead to the development of new treatment strategies. View Publication